Fluorophore(s) to make a "reference solution"

[Emmanuel Levy]

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Hi,

We want to make a "reference solution" that will be imaged each time the
microscope is being used. The purpose is two fold:
- make different experiments comparable in terms of absolute intensities
- monitor laser intensity stability over weeks

We work with multiwell glass bottom plates so a solution would be easy to
use (e.g., we can spike it in an empty well)

We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that can
be mixed together and are stable in the same buffer.

Importantly, they should be stable at room temperature so we could leave
the solution next to the microscope for everyday use. Also, water is
preferable over organic solvents for compatibility with hardware autofocus.

If you have suggestions we'd very much like to hear them.

Thank you,

Emmanuel

[mmodel]

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Hi Emmanuel,


We have done some work on it a while ago. If you use a very concentrated solution of pretty much any fluorophore, its fluorescence would not depend on the depth of the liquid (because light penetrates only into a very small depth) and they practically don't bleach for the same reason. We used Na fluorescein in water for green fluorescence, Rose Bengal for red (it is bright in water and still brighter in DMSO) and acid blue 9 for far red.


Model MA. 2014 (updated from 2002, 2006). Intensity calibration and shading correction for fluorescence microscopes. Curr Protocols in Cytometry. Unit 10-14.

Model MA, Blank JL. 2006. Intensity calibration of a laser scanning confocal microscope based on concentrated dyes. Analyt Quant Cytol Histol. 28:253-261.

Model MA, Burkhardt JK. 2001. A standard for shading correction and calibration in fluorescence microscopy.  Cytometry. 44:309-316.


Mike



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Emmanuel Levy <[hidden email]>
Sent: Saturday, January 28, 2017 7:48 AM
To: [hidden email]
Subject: Fluorophore(s) to make a "reference solution"

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Hi,

We want to make a "reference solution" that will be imaged each time the
microscope is being used. The purpose is two fold:
- make different experiments comparable in terms of absolute intensities
- monitor laser intensity stability over weeks

We work with multiwell glass bottom plates so a solution would be easy to
use (e.g., we can spike it in an empty well)

We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that can
be mixed together and are stable in the same buffer.

Importantly, they should be stable at room temperature so we could leave
the solution next to the microscope for everyday use. Also, water is
preferable over organic solvents for compatibility with hardware autofocus.

If you have suggestions we'd very much like to hear them.

Thank you,

Emmanuel

[Alfred Bahnson]

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We routinely used Inspeck™ fluorescent beads for evaluating set-up and
performance of the fluorescence system from one experiment to the next, but
also for monitoring focus and short term variation in lamp intensity during
experiments.

https://www.google.com/url?sa=t&source=web&rct=j&url=https://tools.thermofisher.com/content/sfs/manuals/mp07219.pdf&ved=0ahUKEwiD9JDsiOXRAhXHQCYKHUIqC8QQFggfMAI&usg=AFQjCNGTRD4O2xorrM-RKPMEPQN97WYsnQ&sig2=dw3zajTMiDjT9v-emNRTGw


These beads (formerly from Molecular Probes, now Thermo-Fisher) come in
many colors and a few sizes and each color comes in a range of calibrated
bead intensities from very bright down to a region close to our detection
level.  If we could get clean images of this lowest-level bead, we knew the
system was near optimal at the start of an experiment.

Typically we used the lowest and next-to-lowest levels in two wells of
every plate and included one or a few sites from each of these wells along
with the live-cell-containing wells to image throughout every multiwell
experiment.

The old mercury arc lamps were too variable for doing much in the way of
normalization of intensities from one scan to the next across whole
experiments,  but the X-cite and Xenon lamps varied slowly enough that the
beads could be used for normalizing this way.  Focus is important for
maintaining consistent intensity, of course.

If you maintain the sterility of your stock, you can add beads directly to
your cell-containg wells for intra-image referencing of focus and
intensity.

Keep in mind that the absorption/emission maxima don't exactly match your
fluorescent compounds, but the different colors are often helpful for
getting oriented with your filters and the nature of your image processing
strategies.

The beads come with an expiration date, but we have no evidence for loss of
intensity from long term exposures or storage.

Another approach using Schott glass has been pioneered by Mike Halter at
NIST:

https://www.google.com/url?sa=t&source=web&rct=j&url=http://ws680.nist.gov/publication/get_pdf.cfm%3Fpub_id%3D915188&ved=0ahUKEwjfneuxkOXRAhVM7CYKHb6mCZMQFggmMAA&usg=AFQjCNGZXmaQbkqxnCVU4l4LsBUpTu6mHg&sig2=XT7LRtXNCQlz6uuo9LFTxA

Hope this helps.

Al Bahnson, partner
Kairos Instruments, LLC

On Jan 28, 2017 7:50 AM, "Emmanuel Levy" <[hidden email]> wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi,
>
> We want to make a "reference solution" that will be imaged each time the
> microscope is being used. The purpose is two fold:
> - make different experiments comparable in terms of absolute intensities
> - monitor laser intensity stability over weeks
>
> We work with multiwell glass bottom plates so a solution would be easy to
> use (e.g., we can spike it in an empty well)
>
> We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that can
> be mixed together and are stable in the same buffer.
>
> Importantly, they should be stable at room temperature so we could leave
> the solution next to the microscope for everyday use. Also, water is
> preferable over organic solvents for compatibility with hardware autofocus.
>
> If you have suggestions we'd very much like to hear them.
>
> Thank you,
>
> Emmanuel
>

[Emmanuel Levy]

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Dear Alfred and Michael,

Thank you, your suggestions are very much appreciated!

All the best,

Emmanuel



On 28 January 2017 at 17:15, Alfred Bahnson <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We routinely used Inspeck™ fluorescent beads for evaluating set-up and
> performance of the fluorescence system from one experiment to the next, but
> also for monitoring focus and short term variation in lamp intensity during
> experiments.
>
> https://www.google.com/url?sa=t&source=web&rct=j&url=https:/
> /tools.thermofisher.com/content/sfs/manuals/mp07219.pdf&ved=
> 0ahUKEwiD9JDsiOXRAhXHQCYKHUIqC8QQFggfMAI&usg=AFQjCNGTRD4O2xorrM-
> RKPMEPQN97WYsnQ&sig2=dw3zajTMiDjT9v-emNRTGw
>
>
> These beads (formerly from Molecular Probes, now Thermo-Fisher) come in
> many colors and a few sizes and each color comes in a range of calibrated
> bead intensities from very bright down to a region close to our detection
> level.  If we could get clean images of this lowest-level bead, we knew the
> system was near optimal at the start of an experiment.
>
> Typically we used the lowest and next-to-lowest levels in two wells of
> every plate and included one or a few sites from each of these wells along
> with the live-cell-containing wells to image throughout every multiwell
> experiment.
>
> The old mercury arc lamps were too variable for doing much in the way of
> normalization of intensities from one scan to the next across whole
> experiments,  but the X-cite and Xenon lamps varied slowly enough that the
> beads could be used for normalizing this way.  Focus is important for
> maintaining consistent intensity, of course.
>
> If you maintain the sterility of your stock, you can add beads directly to
> your cell-containg wells for intra-image referencing of focus and
> intensity.
>
> Keep in mind that the absorption/emission maxima don't exactly match your
> fluorescent compounds, but the different colors are often helpful for
> getting oriented with your filters and the nature of your image processing
> strategies.
>
> The beads come with an expiration date, but we have no evidence for loss of
> intensity from long term exposures or storage.
>
> Another approach using Schott glass has been pioneered by Mike Halter at
> NIST:
>
> https://www.google.com/url?sa=t&source=web&rct=j&url=http://
> ws680.nist.gov/publication/get_pdf.cfm%3Fpub_id%3D915188&ved=
> 0ahUKEwjfneuxkOXRAhVM7CYKHb6mCZMQFggmMAA&usg=
> AFQjCNGZXmaQbkqxnCVU4l4LsBUpTu6mHg&sig2=XT7LRtXNCQlz6uuo9LFTxA
>
> Hope this helps.
>
> Al Bahnson, partner
> Kairos Instruments, LLC
>
> On Jan 28, 2017 7:50 AM, "Emmanuel Levy" <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi,
> >
> > We want to make a "reference solution" that will be imaged each time the
> > microscope is being used. The purpose is two fold:
> > - make different experiments comparable in terms of absolute intensities
> > - monitor laser intensity stability over weeks
> >
> > We work with multiwell glass bottom plates so a solution would be easy to
> > use (e.g., we can spike it in an empty well)
> >
> > We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that
> can
> > be mixed together and are stable in the same buffer.
> >
> > Importantly, they should be stable at room temperature so we could leave
> > the solution next to the microscope for everyday use. Also, water is
> > preferable over organic solvents for compatibility with hardware
> autofocus.
> >
> > If you have suggestions we'd very much like to hear them.
> >
> > Thank you,
> >
> > Emmanuel
> >
>

[Steffen Dietzel]

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Emmanuel,

two thoughts of theoretical nature (since I have no experience with it):

For monitoring laser stability, in your suggested setup you have three
variables: Laser intensity, amount of fluorochrome and the detector
sensitivity. If you would use transmission or reflection of the
excitation laser you would have only two parameters to deal with. This
should do away with the need of a fluorescent solution, avoiding
problems due to fluctuating quality of the fluorescent dye solution,
provided you can use the same detector for reflection and fluorescence.
I haven't done this in long term controls myself though, so maybe others
want to comment.

Second, if you use water, you will have to deal with evaporation which
would change fluorochrome density. Plus, bacterial growth if you keep it
long term. Something non-evaporating with an Ri near water should work,
mixed with dyes. What comes to mind is the Zeiss Immersol W, an
immersion oil with Ri of 1.33. According to the MSDS this is 50-60%
"Dihydroxypropanoxy-methyl derivative of perfluoropolyoxyalcane". But I
have never worked with it, I don't even know if it is hydro- or
lipophilic and I only assume that it does not show evaporation.

Good luck

Steffen


Am 28.01.2017 um 13:48 schrieb Emmanuel Levy:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi,
>
> We want to make a "reference solution" that will be imaged each time the
> microscope is being used. The purpose is two fold:
> - make different experiments comparable in terms of absolute intensities
> - monitor laser intensity stability over weeks
>
> We work with multiwell glass bottom plates so a solution would be easy to
> use (e.g., we can spike it in an empty well)
>
> We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that can
> be mixed together and are stable in the same buffer.
>
> Importantly, they should be stable at room temperature so we could leave
> the solution next to the microscope for everyday use. Also, water is
> preferable over organic solvents for compatibility with hardware autofocus.
>
> If you have suggestions we'd very much like to hear them.
>
> Thank you,
>
> Emmanuel
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Craig Brideau]

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How do you deal with the inter filtering effect of concentrated dye
solutions?

Craig

On Jan 28, 2017 5:31 AM, "MODEL, MICHAEL" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Emmanuel,
>
>
> We have done some work on it a while ago. If you use a very concentrated
> solution of pretty much any fluorophore, its fluorescence would not depend
> on the depth of the liquid (because light penetrates only into a very small
> depth) and they practically don't bleach for the same reason. We used Na
> fluorescein in water for green fluorescence, Rose Bengal for red (it is
> bright in water and still brighter in DMSO) and acid blue 9 for far red.
>
>
> Model MA. 2014 (updated from 2002, 2006). Intensity calibration and
> shading correction for fluorescence microscopes. Curr Protocols in
> Cytometry. Unit 10-14.
>
> Model MA, Blank JL. 2006. Intensity calibration of a laser scanning
> confocal microscope based on concentrated dyes. Analyt Quant Cytol Histol.
> 28:253-261.
>
> Model MA, Burkhardt JK. 2001. A standard for shading correction and
> calibration in fluorescence microscopy.  Cytometry. 44:309-316.
>
>
> Mike
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Emmanuel Levy <[hidden email]>
> Sent: Saturday, January 28, 2017 7:48 AM
> To: [hidden email]
> Subject: Fluorophore(s) to make a "reference solution"
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%
> 3Dconfocalmicroscopy&data=01%7C01%7Cmmodel%40KENT.EDU%
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> Post images on https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cmmodel%40KENT.EDU%
> 7C9f3faac9562b42b975d208d4477c51c1%7Ce5a06f4a1ec44d018f73e7dd15f2
> 6134%7C1&sdata=Eexm0rQGeinb9w5%2FUKLLHG%2BR%2BB81N84TFcCKHZK6aS8%3D&
> reserved=0 and include the link in your posting.
> *****
>
> Hi,
>
> We want to make a "reference solution" that will be imaged each time the
> microscope is being used. The purpose is two fold:
> - make different experiments comparable in terms of absolute intensities
> - monitor laser intensity stability over weeks
>
> We work with multiwell glass bottom plates so a solution would be easy to
> use (e.g., we can spike it in an empty well)
>
> We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that can
> be mixed together and are stable in the same buffer.
>
> Importantly, they should be stable at room temperature so we could leave
> the solution next to the microscope for everyday use. Also, water is
> preferable over organic solvents for compatibility with hardware autofocus.
>
> If you have suggestions we'd very much like to hear them.
>
> Thank you,
>
> Emmanuel
>

[mmodel]

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We actually make use of it. You can prepare a solution where all the excitation light is absorbed within 0.1 um or so. So essentially you have a fixed-depth fluorescent layer, so the total depth of the liquid doesn't matter. And diffusion rapidly replaces photobleached molecules with fresh ones.

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Monday, January 30, 2017 10:59 AM
To: [hidden email]
Subject: Re: Fluorophore(s) to make a "reference solution"

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How do you deal with the inter filtering effect of concentrated dye solutions?

Craig

On Jan 28, 2017 5:31 AM, "MODEL, MICHAEL" <[hidden email]> wrote:

> *****
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> umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Cmmodel
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> 3D&reserved=0 Post images on
> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cmmodel%40KENT.EDU%7C52360c6f34b44adbd3c908d449292780%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=8uRgD0pj6qHZIrp5yvCiQXWOlunZaSyQas3HHkc1y2I%3D&reserved=0 and include the link in your posting.
> *****
>
> Hi Emmanuel,
>
>
> We have done some work on it a while ago. If you use a very
> concentrated solution of pretty much any fluorophore, its fluorescence
> would not depend on the depth of the liquid (because light penetrates
> only into a very small
> depth) and they practically don't bleach for the same reason. We used
> Na fluorescein in water for green fluorescence, Rose Bengal for red
> (it is bright in water and still brighter in DMSO) and acid blue 9 for far red.
>
>
> Model MA. 2014 (updated from 2002, 2006). Intensity calibration and
> shading correction for fluorescence microscopes. Curr Protocols in
> Cytometry. Unit 10-14.
>
> Model MA, Blank JL. 2006. Intensity calibration of a laser scanning
> confocal microscope based on concentrated dyes. Analyt Quant Cytol Histol.
> 28:253-261.
>
> Model MA, Burkhardt JK. 2001. A standard for shading correction and
> calibration in fluorescence microscopy.  Cytometry. 44:309-316.
>
>
> Mike
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Emmanuel Levy <[hidden email]>
> Sent: Saturday, January 28, 2017 7:48 AM
> To: [hidden email]
> Subject: Fluorophore(s) to make a "reference solution"
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%
> 3Dconfocalmicroscopy&data=01%7C01%7Cmmodel%40KENT.EDU%
> 7C9f3faac9562b42b975d208d4477c51c1%7Ce5a06f4a1ec44d018f73e7dd15f2
> 6134%7C1&sdata=3UUstla90o4vCIcGnykUNAbittLNECidk5GpgwTnFcw%3D&reserved
> =0 Post images on https://na01.safelinks.protection.outlook.com/?url=
> http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cmmodel%40KENT.EDU%
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> 6134%7C1&sdata=Eexm0rQGeinb9w5%2FUKLLHG%2BR%2BB81N84TFcCKHZK6aS8%3D&
> reserved=0 and include the link in your posting.
> *****
>
> Hi,
>
> We want to make a "reference solution" that will be imaged each time
> the microscope is being used. The purpose is two fold:
> - make different experiments comparable in terms of absolute
> intensities
> - monitor laser intensity stability over weeks
>
> We work with multiwell glass bottom plates so a solution would be easy
> to use (e.g., we can spike it in an empty well)
>
> We're thus looking for 3 fluorophores (for the 400/488/561 lasers)
> that can be mixed together and are stable in the same buffer.
>
> Importantly, they should be stable at room temperature so we could
> leave the solution next to the microscope for everyday use. Also,
> water is preferable over organic solvents for compatibility with hardware autofocus.
>
> If you have suggestions we'd very much like to hear them.
>
> Thank you,
>
> Emmanuel
>

[Emmanuel Levy]

*****
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Dear Steffen,

Thanks for these suggestions, I very much like the idea of using something
that is not water but has the same refractive index. The reflection idea
sounds great but practically I'm not sure if there are reflective liquids
that are practical to use.

All the best,

Emmanuel

On 30 January 2017 at 10:47, Steffen Dietzel <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Emmanuel,
>
> two thoughts of theoretical nature (since I have no experience with it):
>
> For monitoring laser stability, in your suggested setup you have three
> variables: Laser intensity, amount of fluorochrome and the detector
> sensitivity. If you would use transmission or reflection of the excitation
> laser you would have only two parameters to deal with. This should do away
> with the need of a fluorescent solution, avoiding problems due to
> fluctuating quality of the fluorescent dye solution, provided you can use
> the same detector for reflection and fluorescence. I haven't done this in
> long term controls myself though, so maybe others want to comment.
>
> Second, if you use water, you will have to deal with evaporation which
> would change fluorochrome density. Plus, bacterial growth if you keep it
> long term. Something non-evaporating with an Ri near water should work,
> mixed with dyes. What comes to mind is the Zeiss Immersol W, an immersion
> oil with Ri of 1.33. According to the MSDS this is 50-60%
> "Dihydroxypropanoxy-methyl derivative of perfluoropolyoxyalcane". But I
> have never worked with it, I don't even know if it is hydro- or lipophilic
> and I only assume that it does not show evaporation.
>
> Good luck
>
> Steffen
>
>
> Am 28.01.2017 um 13:48 schrieb Emmanuel Levy:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Hi,
>>
>> We want to make a "reference solution" that will be imaged each time the
>> microscope is being used. The purpose is two fold:
>> - make different experiments comparable in terms of absolute intensities
>> - monitor laser intensity stability over weeks
>>
>> We work with multiwell glass bottom plates so a solution would be easy to
>> use (e.g., we can spike it in an empty well)
>>
>> We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that
>> can
>> be mixed together and are stable in the same buffer.
>>
>> Importantly, they should be stable at room temperature so we could leave
>> the solution next to the microscope for everyday use. Also, water is
>> preferable over organic solvents for compatibility with hardware
>> autofocus.
>>
>> If you have suggestions we'd very much like to hear them.
>>
>> Thank you,
>>
>> Emmanuel
>>
>>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Emmanuel,

On a point scanning confocal microscope you can image at the coverglass
both:

* reflection wavelength ... careful not to 'hurt' the PMT by using
modest laser power, low PMT gain, pinhole can be less than 1.0.

* Michael Model's high concentration fluorophore(s).

* (optional) Constallation and/or other fluorescent beads to add more
structure.

Most coverglasses have crud on them, so reflection image will not be
uniform. You can scratch the glass with a diamond scribe or abrasive (or
write with a Sharpie marker ... most colors fluoresce).

George

p.s. for cell imaging, reflection imaging of the substratum-cell
interface gives excellent interference reflection microscopy (IRM). Can
be quantified to nanometer distances of the underside of the cell. Also
applies to fluorescent beads attached to the sourcface (may want a thin
layer of poly-lysine to facilitate adhesion). Beads often used as
calibration standards for qIRM. Good entry point:

Curr Protoc Cell Biol. 2009 Dec;Chapter 4:Unit 4.23. doi:
10.1002/0471143030.cb0423s45.
Interference reflection microscopy.
Barr VA1, Bunnell SC.
Author information
1National Cancer Institute, National Institutes of Health, Bethesda,
Maryland, USA.
Abstract
Interference reflection microscopy (IRM) is an optical technique used to
study cell adhesion or cell mobility on a glass coverslip. The
interference of reflected light waves generates images with high
contrast and definition. IRM can be used to examine almost any cell that
will rest upon a glass surface, although it is most useful in examining
sites of close contact between a cell and substratum. This unit presents
methods for obtaining IRM images of cells with particular emphasis on
IRM imaging with a laser scanning confocal microscope (LSCM), as most
LSCM are already capable of recording these images without any
modification of the instrument. Techniques are presented for imaging
fixed and live cells, as well as simultaneous multi-channel capture of
fluorescence and reflection images.
Copyright 2009 by John Wiley & Sons, Inc.
PMID: 20013754 PMCID: PMC2824538 DOI: 10.1002/0471143030.cb0423s45


On 1/31/2017 2:15 AM, Emmanuel Levy wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Steffen,
>
> Thanks for these suggestions, I very much like the idea of using something
> that is not water but has the same refractive index. The reflection idea
> sounds great but practically I'm not sure if there are reflective liquids
> that are practical to use.
>
> All the best,
>
> Emmanuel
>
> On 30 January 2017 at 10:47, Steffen Dietzel <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Emmanuel,
>>
>> two thoughts of theoretical nature (since I have no experience with it):
>>
>> For monitoring laser stability, in your suggested setup you have three
>> variables: Laser intensity, amount of fluorochrome and the detector
>> sensitivity. If you would use transmission or reflection of the excitation
>> laser you would have only two parameters to deal with. This should do away
>> with the need of a fluorescent solution, avoiding problems due to
>> fluctuating quality of the fluorescent dye solution, provided you can use
>> the same detector for reflection and fluorescence. I haven't done this in
>> long term controls myself though, so maybe others want to comment.
>>
>> Second, if you use water, you will have to deal with evaporation which
>> would change fluorochrome density. Plus, bacterial growth if you keep it
>> long term. Something non-evaporating with an Ri near water should work,
>> mixed with dyes. What comes to mind is the Zeiss Immersol W, an immersion
>> oil with Ri of 1.33. According to the MSDS this is 50-60%
>> "Dihydroxypropanoxy-methyl derivative of perfluoropolyoxyalcane". But I
>> have never worked with it, I don't even know if it is hydro- or lipophilic
>> and I only assume that it does not show evaporation.
>>
>> Good luck
>>
>> Steffen
>>
>>
>> Am 28.01.2017 um 13:48 schrieb Emmanuel Levy:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> Hi,
>>>
>>> We want to make a "reference solution" that will be imaged each time the
>>> microscope is being used. The purpose is two fold:
>>> - make different experiments comparable in terms of absolute intensities
>>> - monitor laser intensity stability over weeks
>>>
>>> We work with multiwell glass bottom plates so a solution would be easy to
>>> use (e.g., we can spike it in an empty well)
>>>
>>> We're thus looking for 3 fluorophores (for the 400/488/561 lasers) that
>>> can
>>> be mixed together and are stable in the same buffer.
>>>
>>> Importantly, they should be stable at room temperature so we could leave
>>> the solution next to the microscope for everyday use. Also, water is
>>> preferable over organic solvents for compatibility with hardware
>>> autofocus.
>>>
>>> If you have suggestions we'd very much like to hear them.
>>>
>>> Thank you,
>>>
>>> Emmanuel
>>>
>>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München
>> Biomedical Center (BMC)
>> Head of the Core Facility Bioimaging
>>
>> Großhaderner Straße 9
>> D-82152 Planegg-Martinsried
>> Germany
>>
>> http://www.bioimaging.bmc.med.uni-muenchen.de
>>

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650